Sunday 18 November 2007

RNA isolation protocol (1)

RNA isolation by the rapid Guanidinium method
Materials

Mononuclear cell pellet isolated from 1 to 2 ml bone marrow or 5 ml whole blood
HBSS without Ca or Mg
RNA isolation solution I (25 g guanidinium thiocyanate (4 M final), 29.3 ml H2O, 1.76 ml 0.75 M sodium citrate, pH7.0 (25 mM final) , 2.64 ml 10% (w/v) N-lauroylsarcosine (0.5% final) dissolve at 65 degree celcius, store at room temperature (stable > or equal 3 months)
Do not inhale quanidinium thiocyanate and wear gloves when handling it, Do not expose guanidinium thiocyanate to acid conditions, because this may produce cyanide gas.
2 M sodium acetate, pH 4
Buffered phenol
24:1 (v/v) chloroform/isoamyl alcohol
100% and 70% (v/v) ethanol
RNA isolation solution II (10 ml RNA isolation solution I, 72 microlitre 2-mercaptoethanol (0.1 M final) Make fresh before each extraction)
DEPC-treated H2O

5 comments:

Dr Prasit Phowthongkum said...

1. Wash mononuclear cell pellete once with HBSS without Ca or Mg. Resuspened cells in HBSS and divide among three 1.54 ml microcentrifuge tubes. Microcentrifuge 2 min at 100 X g to pellet cells. Discard supernatant. Use immediately or store < or equal 4 months at -70 degree celcius. Use one tube to prepare RNA and the other two repeat assay.

Dr Prasit Phowthongkum said...

2. Add 0.5 ml RNA isolation solution I to cell pellet. Disrupt cells by repeated aspiration. Add 0.05 ml of 2 M sodium acetate. Vortex and microcentrifuge briefly at maximum speed. Add 0.5 ml buffered phenol. Vortex and microcentrifuge. Add 0.1 ml of 24:1 chloroform/isoamyl alcohol. Vortex and microcentrifuge 15 min at maximum speed 4 degree celcius.

Dr Prasit Phowthongkum said...

3. remove 0.4 ml of aqueous (upper) phase to a clean 1.5 ml tube that contains 0.8 ml of 100% ethanol, taking care not to disturb interface. Incubate > 1 hr at -20 degree celcius. Microcentrifuge 15 min at maximum speed, 4 degree celcius. Discard supernatant.

Dr Prasit Phowthongkum said...

4. Add 300 microlitre RNA isolation solution II to pellet and vortex to suspend. Microcentrifuge briefly at maximum speed. Add 600 microlitre of 100% ethanol and mix. Incubate > or equal 1 hr at -20 degree celcius. Microcentrifuge 15 min at high speed, 4 degree celcius. Discard supernatant. Wash pellet with 0.5 ml of 70% ethanol. Discard ethanol. If pellet dislodges from bottom of tube, microcentrifuge 5 min at high speed, 4 degree celcius before removing ethanol.

Dr Prasit Phowthongkum said...

5. Cover open tubes with Paraffin and pierce several times with a fine needle. Dry pellet briefly under vacuum or dry on bench. Store dry RNA < or equal 2 months at – 70 degree celcius or below, or dissolve in 20 microlitre DEPC-treated water if the assay will carried out immediately.